Prostaglandin endoperoxide synthase (PGHS) catalyzes the committed step in the conversion of arachidonic acid to prostaglandins and thromboxane. Prostanoids are "local" hormones which have physiologically important roles in virtually all mammalian tissues. PGHS exhibits both a cyclooxygenase activity involved in the synthesis of PGG2 from arachidonate and a peroxidase activity involved in the reduction of PGG2 to PGH2. The cyclooxygenase activity of PGHS is a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, information about how the enzyme functions and how it interacts with NSAIDs has potential therapeutic importance. The long-range goal of our research is to determine the mechanisms of cyclooxygenase and peroxidase catalysis. Current work is concentrated on identifying amino acids essential for catalysis by, PGHS. During the past budget period, histidine and tyrosine residues essential for cyclooxygenase and peroxidase activities were identified. In addition, the mechanism by which aspirin inhibits cyclooxygenase activity was determined. Finally, the sites of N-glycosylation of PGHS-1 were identified, and the importance of N-glycosylation in the expression of PGHS activity was established. We now propose: (a) to determine if the tyrosyl radical observed during catalysis initiates suicide inactivation of cyclooxygenase activity and to identify the tyrosine residue which is the source of the radical; these studies will involve a combination of site-directed mutagenesis and EPR spectroscopy. (b) to characterize more completely the structure of the cyclooxygenase active site; photoaffinity probes will be employed to identify active site residues; in addition, chimeras of murine PGHS-1 and PGHS-2 isozymes will be used to determine the importance of the C-terminal domain, encoded by the last exon and containing the aspirin acetylation site, in governing isozyme specificities toward NSAIDs; in addition, in vitro expression systems will be utilized to determine the specificities of the human PGHS-1 and -2 isozymes toward NSAIDs. (c) to determine the inside/outside orientation of various domains of PGH synthase within the endoplasmic reticulum; these studies Will employ a combination of several anti-peptide antibodies and a photoactive probe reactive with amino acids located in transmembrane regions.